Synthetic metabolic pathways for conversion of CO₂ into secreted short-to medium-chain hydrocarbons using cyanobacteria

Abstract

The objective of this study was to implement direct sunlight-driven conversion of CO2 into a naturally excreted ready-to-use fuel. We engineered four different synthetic metabolic modules for biosynthesis of short-to medium-chain length hydrocarbons in the model cyanobacterium Synechocystis sp. PCC 6803. In module 1, the combination of a truncated clostridial n-butanol pathway with over-expression of the native cyanobacterial aldehyde deformylating oxygenase resulted in small quantities of propane when cultured under closed conditions. Direct conversion of CO2 into propane was only observed in strains with CRISPRi-mediated repression of three native putative aldehyde reductases. In module 2, three different pathways towards pentane were evaluated based on the polyunsaturated fatty acid linoleic acid as an intermediate. Through combinatorial evaluation of bioreaction ingredients it was concluded that linoleic acid undergoes a spontaneous non-enzymatic reaction to yield pentane and hexanal. When Synechocystis was added to the bioreaction, hexanal was converted into 1-hexanol, but there was no further stimulation of pentane biosynthesis. For modules 3 and 4, several different acyl-ACP thioesterases were evaluated in combination with two different decarboxylases. Small quantities of 1-heptene and 1-nonene were observed in strains expressing the desaturase-like enzyme UndB from Pseudomonas mendocina in combination with C8-C10 preferring thioestersaes. When UndB instead was combined with a C12-specific ‘UcFatB1 thioesterase, this resulted in ten-fold increase of alkene biosynthesis. When UndB was replaced with the light-dependent FAP decarboxylase, both undecane and tridecane accumulated, albeit with a 10-fold drop in productivity. Optimization of the RBS, promoter and gene order in these synthetic operons resulted in 1-alkene bioproductivity of 230 mg/L after 10 d with 15% carbon partitioning. In conclusion, the direct bioconversion of CO2 into secreted and ready-to-use hydrocarbon fuel was accomplished and optimal results were obtained with UndB and a C12 chain-length specific thioesterase.HighlightsMultiple repression of endogenous aldehyde reductases/dehydrogenases by CRISPRi enabled propane biosynthesisBiosynthesis of short-medium chain hydrocarbons (C7-C11) in a cyanobacterium was demonstrated for the first timeThe final enzymes of the hydrocarbon pathways influenced both productivity and product profileAll volatile products were naturally secreted and accumulated outside of the cell